Monoclonal antibodies (MAbs) of the immunoglobulin G (IgG) type are an important class of therapeutic glycoproteins. Compelling evidence has indicated that the fine structures of the glycans at the conserved N-glycosylation site (Asn-297) of the Fc domain are responsible for the distinct effector functions of MAbs, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and activation of apoptosis. In addition, a special sialylated Fc glycoform was identified to be responsible for the antiinflammatory activity of intravenous immunoglobulin (IVIG). However, progress in understanding the functional roles of IgG-Fc glycosylation is hampered by the tremendous structural heterogeneity of Fc domain glycans. In addition, controlling glycosylation of MAbs in expression to a desired homogeneous glycoform is still a challenging task. In this application, we propose to explore a chemoenzymatic method to make a library of homogeneously glycosylated IgG-Fc and selected glycoforms of MAbs. Through performing Fc receptor binding studies, we aim to understand how different glycan structures can fine tune the effector functions of IgG and IgG-Fc protein. We have performed important preliminary studies indicating that it is feasible to use the endoglycosidase-based transglycosylation approach to construct defined, homogeneous glycoforms of human IgG-Fc. Building on this success, we propose to pursue three specific aims. Aim 1 is to explore a chemoenzymatic method for the construction of various pure glycoforms of IgG-Fc. Aim 2 is to evaluate the structure-activity realtionships of different Fc domain glycoforms in Fc receptor binding, and to evaluate ADCC activity of selectively glycoengineered monoclonal antibodies. Aim 3 is to synthesize novel Fc domain glycoforms for evaluating the roles of IgG-Fc glycosylation in anti-inflammatory activity. The knowledge gained from the proposed research will eventually facilitate the development of novel glycoforms of MAbs and IgG-Fc proteins as effective therapeutics.